Method for measuring calcium 6S-5-methyltetrahydrofolate in milk system through high performance liquid chromatography

Abstract

The invention discloses a method for determining calcium 6S-5-methyltetrahydrofolate in a milk system through high performance liquid chromatography, experiments prove that degradation of (6S)-5-MTHF-Ca can be blocked and the stability can be improved by jointly using two reducing substances in a sample treatment process, so that the recovery rate of the (6S)-5-MTHF-Ca is improved, and the method is suitable for industrial production. The average recovery rate of the (6S)-5-MTHF-Ca enhanced modified milk is 97.8%, the RSD is 0.56%, the average recovery rate of the (6S)-5-MTHF-Ca enhanced milk powder is 97.9%, the RSD is 0.42%, the recovery rates in CN116718714A in the prior art are 85.57%, 86.78% and 84.78%, the average recovery rate is 85.81%, and the RSD is 1.17%, and obviously, the recovery rate of the method is obviously higher than that of a contrast file and reaches the optimal value.

Description

Technical Field

[0001] The present invention belongs to the technical field of milk system detection, and particularly relates to a method for determining calcium 6S-5-methyltetrahydrofolate in a milk system by high performance liquid chromatography. Background Art

[0002] (6S)-5-Methyltetrahydrofolate calcium (C) crystal form ((6S)-5-MTHF-Ca), namely naturalized (active) folic acid, is an upgrade of the production process on the basis of the original active folic acid: removing processing aids such as heavy metal catalysts, formaldehyde and benzenesulfonic acid that are harmful to embryonic development, and strictly controlling impurities with potential safety hazards in JK12A. The product not only has the structure of natural folic acid but also has the safety of natural folic acid. At the same time, the (6S)-5-methyltetrahydrofolate calcium (C) crystal form has excellent stability.

[0003] In China, the application of (6S)-5-methyltetrahydrofolate calcium (C) crystal form is still in its infancy, and the common product forms on the market still remain at dietary supplements. Milk contains rich nutritional components and bioactive components. Strengthening the milk system with (6S)-5-MTHF-Ca can achieve the purpose of supplementing folic acid and at the same time meet the human body's needs for a variety of nutrients.

[0004] At present, a systematic detection method for (6S)-5-MTHF-Ca in a milk system has not been established. Compared with the microbiological method for determining folic acid content in dairy products in existing patents, the high performance liquid chromatography method is simple to operate, easy to master, has higher stability and stronger accuracy. Patent publication number CN116718714A is applicable to the detection of calcium 6S-5-methyltetrahydrofolate in cosmetics, and its main recovery rates are 85.57%, 86.78%, 84.78%, and the average recovery rate is 85.81%. The determination method of precision is not disclosed; this patent is applicable to the detection in a milk system. Compared with CN116718714A, a protective agent is added during the sample pretreatment process in this patent. Two protective agents are jointly used in this patent to better avoid oxidative degradation, reduce the sampling amount, and change the extraction agents n-butanol and chloroform to non-toxic acetic acid, making the sample pretreatment process safer and more effective, and the final recovery rate is also greatly improved. At the same time, a precision determination method is given. Therefore, the method for determining (6S)-5-MTHF-Ca in a milk system by high performance liquid chromatography is innovative and will play a role in corresponding food detection in the future. Summary of the Invention

[0005] The purpose of the present invention is to design a method for determining calcium (6S)-5-methyltetrahydrofolate in milk systems by high performance liquid chromatography. In this method, the average recovery rate of (6S)-5-MTHF-Ca fortified modulated milk is 97.8%, and the RSD is 0.56%. The average recovery rate of (6S)-5-MTHF-Ca fortified milk powder is 97.9%, and the RSD is 0.42%. The method for determining precision is also given.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for determining calcium (6S)-5-methyltetrahydrofolate in milk systems by high performance liquid chromatography, characterized in that the steps of the method are as follows: S1: During the sample pretreatment process, two reducing substances are added to the fortified dairy product solution to further improve the recovery rate of (6S)-5-MTHF-Ca; S2: Standard treatment solution 6S-A of (6S)-5-MTHF-Ca fortified dairy products: Weigh 0.05 g of 6S-5-MTHF-Ca, dissolve it ultrasonically with an appropriate amount of liquid milk, place it in a brown volumetric flask, and dilute it to the scale with liquid milk. The concentration is 200 μg / mL; S3: Respectively pipette 0.10 mL to 5.00 mL of 6S-A into a 100 mL brown volumetric flask, and dilute it to the scale with liquid milk. The concentration of the standard solution is 0.2 to 10.0 μg / mL; S4: Respectively pipette 5 mL of the standard solution into centrifuge tubes, after treating according to the pretreatment method, analyze and determine different concentrations of the standard solution by HPLC method; S5: Determine the equation Y = kX + c according to the concentration of the standard solution and the corresponding peak area, and obtain the standard curve and equation for detecting (6S)-5-MTHF-Ca in the milk system; S6: Test the 0.2 μg / mL standard solution 3 times by HPLC method. The corresponding concentration at a signal-to-noise ratio of 10:1 is the limit of quantification; the corresponding concentration at a signal-to-noise ratio of 3:1 is the limit of detection; S7: Take 5 mL of the sample for sample pretreatment and inject it 5 times, calculate the relative standard deviation of the sample determination, and obtain the precision.

[0007] The specific method in S1 is as follows: Step 1: Preparation of the standard solution: Weigh 0.05 g of DL-5-methyltetrahydrofolate calcium standard DL-5-MTHF-Ca, dissolve it ultrasonically with an appropriate amount of ultrapure water, and dilute it to obtain a standard stock solution with a concentration of 200 μg / mL; Step 2: Accurately pipette 0.1 mL to 4.00 mL of the standard stock solution into a 100 mL brown volumetric flask, and make up to the mark with ultrapure water. The concentration range of the standard solution is 0.2 to 8.0 μg / mL; Step 3: Analyze and determine the standard solutions with different concentrations by HPLC method; Step 4: Determine the equation Y = kX + c based on the concentration of the standard solution and the corresponding peak area to obtain the standard curve and equation; Step 5: Pipette the fortified dairy product solution into a centrifuge tube, and add two reducing substances to the fortified dairy product solution; Step 6: Heat-treat in a water bath at 80 - 85 °C for 25 - 35 min, and cool to room temperature; Step 7: Add 3.2% (v / v) acetic acid solution according to the ratio of sample solution : extractant = 1:1 (v / v); Step 8: Centrifuge at 6000 - 8000 rmp in a centrifuge for 8 - 12 min; take the supernatant; after removing the interference of milk protein, filter through a 0.22 μm filter head and inject into the injection vial; determine the peak area of (6S)-5-MTHF-Ca in the sample solution by HPLC method; substitute into the standard curve of peak area and concentration to calculate the recovery rate of (6S)-5-MTHF-Ca.

[0008] Add two reducing substances to the fortified dairy product solution. Add two reducing substances according to the volume ratio of the fortified dairy product solution of 1:7 to 1:9. The reducing substances include but are not limited to L-ascorbic acid, sodium ascorbate, L / D-cysteine, glutathione, β-mercaptoethanol, dithiothreitol, 2-mercaptoethanol, 3-mercapto-3-methylbutanol.

[0009] Furthermore, the fortified milk powder in S2 must be dissolved with warm water at a ratio of 2% w / v.

[0010] Compared with the existing technology with the publication number CN116718714A, the beneficial effects of the present invention are as follows: It has been experimentally proven in the present invention that the combined use of two reducing substances during the sample treatment process can block the degradation of (6S)-5-MTHF-Ca, improve the stability, and thus improve the recovery rate of (6S)-5-MTHF-Ca. The average recovery rate of (6S)-5-MTHF-Ca fortified milk in the present invention is 97.8%, and the RSD is 0.56%. The average recovery rate of (6S)-5-MTHF-Ca fortified milk powder is 97.9%, and the RSD is 0.42%. In the existing technology CN116718714A, the recovery rates are 85.57%, 86.78%, 84.78%, the average recovery rate is 85.81%, and the RSD is 1.17%. Obviously, the recovery rate of the present invention is significantly higher than that of the comparative document and has reached the optimal value.

[0011] This patent uses high performance liquid chromatography to determine the content of (6S)-5-MTHF-Ca in fortified milk systems, filling the gap in the systematic detection method for (6S)-5-MTHF-Ca in fortified milk systems. At the same time, it also innovates the method for removing proteins in milk systems. The established detection method has high recovery rate and good stability, laying a foundation for the popularization and application of (6S)-5-MTHF-Ca. Description of the Drawings

[0012] Figure 1 It is a flow chart.

[0013] Figure 2 It is a graph showing the relationship between the concentration of the standard solution in Example 1 and the corresponding peak area.

[0014] Figure 3 It is the HPLC chromatogram of the formulated milk in Example 1.

[0015] Figure 4 It is the HPLC chromatogram of the (6S)-5-MTHF-Ca fortified formulated milk in Example 1.

[0016] Figure 5 It is the HPLC chromatogram of the formulated milk powder in Example 2.

[0017] Figure 6 It is the HPLC chromatogram of the (6S)-5-MTHF-Ca fortified formulated milk powder in Example 2.

[0018] Figure 7 It is the graph showing the relationship between the concentration of the standard solution and the corresponding peak area in Example 4 Detailed Description of the Invention

[0019] The present invention will be further described below with reference to the accompanying drawings: As Figure 1 shown, the entire detailed determination method is presented. A specific and detailed method for determining 6S-5-methyltetrahydrofolate calcium in milk systems by high performance liquid chromatography is as follows: 1. Preparation of the standard solution: Weigh 0.05 g of DL-5-methyltetrahydrofolate calcium standard (DL-5-MTHF-Ca), dissolve it in an appropriate amount of ultrapure water by ultrasonic treatment, and then dilute it to obtain a standard stock solution with a concentration of 200 μg / mL.

[0020] 2. Accurately pipette 0.1 mL to 4.00 mL of the standard stock solution into a 100 mL brown volumetric flask, and make up to the mark with ultrapure water. The concentration range of the standard solution is 0.2 to 8.0 μg / mL.

[0021] 3. Analyze and determine the standard solutions with different concentrations by the HPLC method in Appendix A of T / BPSPA 0016—2022.

[0022] 4. Determine the equation Y = kX + c based on the standard solution concentration and the corresponding peak area to obtain the standard curve and equation.

[0023] 5. Pipette the fortified dairy product solution into a centrifuge tube (for fortified milk powder, it must be dissolved in warm water at a ratio of 2% (w / v)). Add two kinds of protective agents according to the volume of the sample solution at a ratio of 1:7 - 1:9. The protective agents include but are not limited to reducing substances such as L-ascorbic acid, sodium ascorbate, L / D-cysteine, glutathione, β-mercaptoethanol, dithiothreitol, 2-mercaptoethanol, 3-mercapto-3-methylbutanol, etc.

[0024] 6. Heat-treat in a water bath at 80 - 85 °C for 25 - 35 min, and cool to room temperature.

[0025] 7. Add 3.2% (v / v) acetic acid solution according to the ratio of sample solution: extractant = 1:1 (v / v).

[0026] 8. Centrifuge in a centrifuge at 6000 - 8000 rmp for 8 - 12 min.

[0027] 9. Take the supernatant.

[0028] 10. After removing the interference of milk proteins, filter through a 0.22 μm filter head and inject into a sample vial.

[0029] 11. Use the HPLC method in Appendix A of T / BPSPA 0016—2022 to determine the peak area of (6S)-5-MTHF-Ca in the sample solution.

[0030] 12. Substitute into the standard curve of peak area and concentration to calculate the recovery rate of (6S)-5-MTHF-Ca.

[0031]

[0032] 13. (6S)-5-MTHF-Ca fortified dairy product standard treatment solution (6S-A): Weigh 0.1 g (accurate to 0.0001 g) of 6S-5-MTHF-Ca, add an appropriate amount of liquid milk, dissolve it by ultrasonic treatment, and place it in a brown volumetric flask. Dilute it to the scale with liquid milk, and the concentration is 200 μg / mL.

[0033] 14. Pipette 0.05 mL - 2.50 mL of 6S-A into 50 mL brown volumetric flasks respectively, and dilute to the scale with liquid milk. The concentration of the standard solution is 0.2 - 10.0 μg / mL.

[0034] 15. Pipette 5 mL of the standard solution into centrifuge tubes respectively. After treating according to the above pretreatment method, analyze and determine the standard solutions with different concentrations by HPLC method.

[0035] 16. Determine the equation Y = kX + c based on the standard solution concentration and the corresponding peak area, and obtain the standard curve and equation for the detection of (6S)-5-MTHF-Ca in the milk system.

[0036] 17. Test the 0.2 μg / mL standard solution by HPLC three times. The corresponding concentration at a signal-to-noise ratio of 10:1 is taken as the limit of quantitation, and the corresponding concentration at a signal-to-noise ratio of 3:1 is taken as the limit of detection.

[0037] 18. Take 5 mL of the sample for sample pretreatment and inject it five times, and calculate the relative standard deviation of the sample determination.

[0038] Example 1 in combination with Figure 3 and Figure 4 Determination of the method recovery rate after pretreatment of (6S)-5-MTHF-Ca fortified modulated milk shown 1. DL-5-methyltetrahydrofolate calcium standard treatment solution (DL-A): Weigh 0.1 g (accurate to 0.0001 g) of DL-5-methyltetrahydrofolate calcium standard (DL-5-MTHF-Ca), dissolve it ultrasonically with an appropriate amount of ultrapure water, place it in a 100 mL brown volumetric flask, and dilute it to the mark to obtain a standard solution with a concentration of 1000 μg / mL, and store it in a refrigerator at 4 °C. 2. 20% DL-5-methyltetrahydrofolate calcium standard treatment solution (DL-20): Accurately pipette 10 mL of DL-A into a 50 mL brown volumetric flask, dilute it to the mark with ultrapure water, and the concentration is 200 μg / mL.

[0039] 3. Accurately pipette 0.05 mL, 0.10 mL, 0.20 mL, 0.40 mL, 0.80 mL, 1.00 mL, 1.50 mL, 2.00 mL of DL-20 into 50 mL brown volumetric flasks, and dilute them to the mark with ultrapure water. The concentrations of the standard solutions are 0.2, 0.4, 0.8, 1.6, 3.2, 4.0, 6.0, 8.0 μg / mL respectively.

[0040] 4. Analyze and determine different concentrations of standard solutions by HPLC method in Appendix A of T / BPSPA 0016—2022.

[0041] 5. Figure 2 As shown, determine the equation Y = 17648X + 379.99 based on the standard solution concentration and the corresponding peak area, and the correlation coefficient R 2 = 0.999.

[0042] Transfer the fortified milk sample solution containing 12 μg / mL of (6S)-5-MTHF-Ca into a centrifuge tube, and add 1% (w / v) ascorbic acid solution and 2% (v / v) dithiothreitol solution according to the volume ratio of the sample solution of 1:9.

[0043] 7. Heat-treat in a water bath at 83.7 ± 0.2 °C for 30 min, and cool to room temperature.

[0044] 8. Add 3.2% (v / v) acetic acid solution according to the ratio of sample solution: extractant (1:1, v / v).

[0045] 9. Centrifuge in a centrifuge at 6000 rmp for 10 min.

[0046] 10. Take the supernatant.

[0047] 11. Filter through a 0.22 μm filter head and inject into an injection vial.

[0048] 12. Use the HPLC method in Appendix A of T / BPSPA 0016—2022 to determine the peak area of (6S)-5-MTHF-Ca in the sample solution.

[0049] 13. Substitute into the standard curve, and calculate that the recovery rate of the sample pretreatment method is 97.8%.

[0050] Example 2 in combination with Figure 5 and 6 as shown, determination of the recovery rate of the pretreatment method for (6S)-5-MTHF-Ca fortified modulated milk powder (fortified milk powder) 1. DL-5-methyltetrahydrofolate calcium standard treatment solution (DL-A): Weigh 0.1 g (accurate to 0.0001 g) of DL-5-methyltetrahydrofolate calcium standard (DL-5-MTHF-Ca), dissolve it in an appropriate amount of ultrapure water by ultrasonic treatment, and place it in a 100 mL brown volumetric flask, and dilute to the scale to obtain a standard solution with a concentration of 1000 μg / mL, and store it in a refrigerator at 4 °C; 2. 20% DL-5-methyltetrahydrofolate calcium standard treatment solution (DL-20): Accurately transfer 10 mL of DL-A into a 50 mL brown volumetric flask, dilute it to the scale with ultrapure water, and the concentration is 200 μg / mL.

[0051] 3. Accurately transfer 0.05 mL, 0.10 mL, 0.20 mL, 0.40 mL, 0.80 mL, 1.00 mL, 1.50 mL, 2.00 mL of DL-20 into 50 mL brown volumetric flasks, and dilute to the scale with ultrapure water. The concentrations of the standard solutions are 0.2, 0.4, 0.8, 1.6, 3.2, 4.0, 6.0, 8.0 μg / mL respectively.

[0052] 4. Analyze and determine standard solution of different concentrations by HPLC method.

[0053] 5. Determine the equation Y = 17648X + 379.99 based on the concentration of the standard solution and the corresponding peak area, with the correlation coefficient R 2 = 0.999.

[0054] 6. Weigh 2 g of (6S)-5-MTHF-Ca fortified milk powder, dissolve it in warm water, and make up the volume to 100 mL in a volumetric flask.

[0055] 7. Pipette the sample solution of (6S)-5-MTHF-Ca fortified milk powder containing 2 μg / mL into a centrifuge tube, and add 1% (w / v) ascorbic acid solution and 2% (v / v) β-mercaptoethanol solution according to the volume ratio of the sample solution of 1:8.

[0056] 8. Heat-treat in a water bath at 83 ± 0.2 °C for 30 min, and cool to room temperature.

[0057] 9. Add 3.2% (v / v) acetic acid solution according to the ratio of sample solution: extractant = 1:1 (v / v).

[0058] 10. Centrifuge at 6000 rmp for 10 min in a centrifuge.

[0059] 11. Take the supernatant.

[0060] 12. Filter through a 0.22 μm filter head and inject into an injection vial. 13. Determine the peak area of (6S)-5-MTHF-Ca in the sample solution by HPLC method in Appendix A of T / BPSPA 0016—2022.

[0061] 14. Substitute into the standard curve of peak area and concentration, and calculate the recovery rate to be 97.9%.

[0062] Example 3 Determination of the recovery rate of the method after the pretreatment of (6S)-5-MTHF-Ca fortified modified milk Drawing of the standard curve: Weigh 0.1 g (accurate to 0.0001 g) of DL-5-methyltetrahydrofolate calcium standard (DL-5-MTHF-Ca), dissolve it in an appropriate amount of ultrapure water by ultrasonic, and place it in a 500 mL brown volumetric flask, and dilute to the scale to prepare a standard stock solution with a concentration of 200 μg / mL.

[0063] Accurately pipette 0.05 mL - 2.00 mL of the standard stock solution into a 50 mL brown volumetric flask, and make up the volume to the scale with ultrapure water. The concentrations of the standard solutions are 0.2 - 8.0 μg / mL respectively.

[0064] The standard solutions with different concentrations were analyzed and determined by HPLC method.

[0065] 4. Determine the equation Y = 17648X + 379.99 based on the concentration of the standard solution and the corresponding peak area, with the correlation coefficient R 2 = 0.999.

[0066] 5. Respectively pipette the (6S)-5-MTHF-Ca fortified milk containing 0.8 μg / mL and 1.2 μg / mL, and the fortified milk powder solutions containing 2 μg / mL and 8 μg / mL into centrifuge tubes, and add 1% (w / v) ascorbic acid solution and 2% (v / v) 2-mercaptoethanol solution according to the volume ratio of the sample solution of 1:8.

[0067] 6. Heat-treat in a water bath at 84 ± 0.2 °C for 30 min, and cool to room temperature.

[0068] 7. Add 3.2% (v / v) acetic acid solution according to the ratio of sample solution: extractant = 1:1 (v / v).

[0069] 8. Centrifuge at 6500 rmp for 10 min in a centrifuge.

[0070] 9. Take the supernatant.

[0071] 10. Filter through a 0.22 μm filter head and inject into the injection vial. 11. Use the HPLC method in Appendix A of T / BPSPA 0016—2022 to determine the peak area of (6S)-5-MTHF-Ca in the sample solution.

[0072] 12. Substitute into the standard curve and calculate the recovery rates to be 97.8% and 97.9%.

[0073] Recovery rate of the method

[0074] After verification by examples, the average recovery rate of (6S)-5-MTHF-Ca fortified modified milk is 97.8% with an RSD of 0.56%; the average recovery rate of (6S)-5-MTHF-Ca fortified milk powder is 97.9% with an RSD of 0.42%. The recovery rates of Patent CN116718714A are 85.57%, 86.78%, and 84.78%, with an average recovery rate of 85.81% and an RSD of 1.17%. The recovery rate of this method has been greatly improved. After verification by examples, through the combined method of water bath heating and efficient protein removal with a small amount of extractant, the protein is effectively removed, facilitating the determination of (6S)-5-MTHF-Ca in the fortified milk system by HPLC method. The recovery rate of (6S)-5-MTHF-Ca is high, and the recovery rates are all above 97%.

[0075] Example 4 Establishment of the standard curve of (6S)-5-MTHF-Ca in milk systems, determination of the limit of quantitation, limit of detection and precision 1. (6S)-5-MTHF-Ca fortified milk standard treatment solution (6S-A): Weigh 0.1 g (accurate to 0.0001 g) of 6S-5-MTHF-Ca, dissolve it ultrasonically in an appropriate amount of liquid milk, place it in a 100 mL brown volumetric flask, and dilute it to the mark to obtain a standard solution with a concentration of 1000 μg / mL, and store it in a refrigerator at 4 °C. (For fortified milk powder, it must be dissolved in warm water at a ratio of 2% (w / v).) 2. 20% (6S)-5-MTHF-Ca fortified milk standard treatment solution (6S-20): Accurately pipette 10 mL of 6S-A into a 50 mL brown volumetric flask, dilute it to the mark with liquid milk, and the concentration is 200 μg / mL.

[0076] 3. Pipette 0.1 mL - 2.50 mL of 6S-20 into 50 mL brown volumetric flasks respectively, dilute it to the mark with liquid milk, and the concentration of the standard solution is 0.4 - 10.0 μg / mL.

[0077] 4. Pipette fortified milk sample solutions with different concentrations into centrifuge tubes respectively, and add 1% (w / v) ascorbic acid solution and 2% (v / v) 2-mercaptoethanol solution according to the volume ratio of the sample solution of 1:8.

[0078] 5. Heat-treat in a water bath at 83.7 °C for 30 min, and cool to 35 °C.

[0079] 6. Add 3.2% (v / v) acetic acid solution according to the ratio of standard solution : extractant = 1:1 (v / v).

[0080] 7. Centrifuge in a centrifuge at 6000 rmp for 10 min.

[0081] 8. Take the supernatant.

[0082] 9. Filter through a 0.22 μm filter head and inject it into an injection vial.

[0083] 10. Use the HPLC method in Appendix A of T / BPSPA 0016—2022 to determine the peak area of (6S)-5-MTHF-Ca in fortified solutions with different concentrations.

[0084] 11. Figure 7 Determine the equation Y = 1714.9X + 298.46 according to the standard solution concentration and the corresponding peak area, and the correlation coefficient R 2 = 0.9993.

[0085] The standard solution of 0.2 µg / mL was tested 3 times by HPLC. The corresponding concentration at a signal-to-noise ratio of 10:1 was taken as the quantitation limit, and the corresponding concentration at a signal-to-noise ratio of 3:1 was taken as the detection limit. The quantitation limit of this method was 0.0736 µg / mL, and the detection limit was 0.0243 µg / mL.

[0086] According to the pretreatment method, the same concentration of milk system sample solution was analyzed and determined by HPLC for [number of times]. The results are shown in the following table.

[0087] Precision

[0088] After example verification, this method has a good linear relationship in the range of 0 - 10 μg / mL, and the reproducibility of the measurement results is good. The relative standard deviation of (6S)-5-MTHF-Ca fortified modulated milk is 0.26%, and the relative standard deviation of (6S)-5-MTHF-Ca fortified milk powder solution is 0.09%. The measurement results are relatively stable, indicating that this method has good precision.

[0089] The above are all preferred embodiments of the present invention. For those of ordinary skill in the art, without departing from the principle of the present invention, the modifications of various equivalent forms of the present invention all fall within the protection scope of the appended claims of this application.

Claims

1. A method for determining calcium 6S-5-methyltetrahydrofolate in milk systems by high performance liquid chromatography, characterized in that: The method steps are as follows: S1: During the sample pretreatment process, two reducing substances are added to the fortified dairy product solution to further improve the recovery rate of (6S)-5-MTHF-Ca; S2: (6S)-5-MTHF-Ca fortified dairy product standard treatment solution 6S-A: Weigh 0.05 g of 6S-5-MTHF-Ca, dissolve it ultrasonically with an appropriate amount of liquid milk, place it in a brown volumetric flask, and dilute it to the scale with liquid milk. The concentration is 200 μg / mL; S3: Respectively pipette 0.10 mL - 5.00 mL of 6S-A into a 100 mL brown volumetric flask, and dilute it to the scale with liquid milk. The concentration of the standard solution is 0.2 - 10.0 μg / mL; S4: Respectively pipette 5 mL of the standard solution into centrifuge tubes. After treating according to the pretreatment method, use HPLC to analyze and determine the standard solutions with different concentrations; S5: Determine the equation Y = kX + c according to the concentration of the standard solution and the corresponding peak area, and obtain the standard curve and equation for the detection of (6S)-5-MTHF-Ca in the milk system; S6: Use the 0.2 μg / mL standard solution and test it 3 times by HPLC. The corresponding concentration at a signal-to-noise ratio of 10:1 is the quantitative limit; the corresponding concentration at a signal-to-noise ratio of 3:1 is the detection limit; S7: Take 5 mL of the sample for sample pretreatment and inject it 5 times, calculate the relative standard deviation of the sample determination, and obtain the precision; 2. The method for determining calcium 6S-5-methyltetrahydrofolate in a milk system by high performance liquid chromatography according to claim 1, wherein: The specific method in S1 is as follows: Step 1: Preparation of the standard solution: Weigh 0.05 g of the DL-5-methyltetrahydrofolate calcium standard DL-5-MTHF-Ca, dissolve it ultrasonically with an appropriate amount of ultrapure water, and dilute it to obtain a standard stock solution with a concentration of 200 μg / mL; Step 2: Accurately pipette 0.1 mL - 4.00 mL of the standard stock solution into a 100 mL brown volumetric flask, and dilute it to the scale with ultrapure water. The concentration range of the standard solution is 0.2 - 8.0 μg / mL; Step 3: Use HPLC to analyze and determine the standard solutions with different concentrations; Step 4: Determine the equation Y = kX + c according to the concentration of the standard solution and the corresponding peak area, and obtain the standard curve and equation; Step 5: Pipette the fortified dairy product solution into a centrifuge tube, and add two reducing substances to the fortified dairy product solution; Step 6: Heat-treat it in a water bath at 80 - 85 °C for 25 - 35 min, and cool it to room temperature; Step 7: Add 3.2% (v / v) acetic acid solution according to the ratio of sample solution ∶ extractant = 1:1 (v / v); Step 8: Place it in a centrifuge and centrifuge at 6000 - 8000 rmp for 8 - 12 min; take the supernatant; after removing the interference of milk proteins, filter it through a 0.22 μm filter head and inject it into the injection vial; Use HPLC to measure the peak area of (6S)-5-MTHF-Ca in the sample solution; substitute it into the standard curve of peak area and concentration, and calculate the recovery rate of (6S)-5-MTHF-Ca.

3. A method for determining calcium 6S-5-methyltetrahydrofolate in a milk system by high performance liquid chromatography according to claim 1 or 2, characterized in that: Two reducing substances are added to the fortified dairy product solution, and the two reducing substances are added in a volume ratio of 1:7 to 1:9 based on the volume of the fortified dairy product solution. The reducing substances include, but are not limited to, L-ascorbic acid, sodium ascorbate, L / D-cysteine, glutathione, β-mercaptoethanol, dithiothreitol, 2-mercaptoethanol, 3-mercapto-3-methylbutanol.

4. A method for determining calcium 6S-5-methyltetrahydrofolate in a milk system by high performance liquid chromatography according to claim 1, characterized in that: The fortified modified milk powder in S2 must be dissolved with warm water at a ratio of 2% w / v.

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